Sub-telomere directed gene expression during initiation of invasive aspergillosis

Abstract

Aspergillus fumigatus is a common mould whose spores are a component of the normal airborne flora. Immune dysfunction permits developmental growth of inhaled spores in the human lung causing aspergillosis, a significant threat to human health in the form of allergic, and life-threatening invasive infections. The success of A. fumigatus as a pathogen is unique among close phylogenetic relatives and is poorly characterised at the molecular level. Recent genome sequencing of several Aspergillus species provides an exceptional opportunity to analyse fungal virulence attributes within a genomic and evolutionary context. To identify genes preferentially expressed during adaptation to the mammalian host niche, we generated multiple gene expression profiles from minute samplings of A. fumigatus germlings during initiation of murine infection. They reveal a highly co-ordinated A. fumigatus gene expression programme, governing metabolic and physiological adaptation, which allows the organism to prosper within the mammalian niche. As functions of phylogenetic conservation and genetic locus, 28% and 30%, respectively, of the A. fumigatus subtelomeric and lineage-specific gene repertoires are induced relative to laboratory culture, and physically clustered genes including loci directing pseurotin, gliotoxin and siderophore biosyntheses are a prominent feature. Locationally biased A. fumigatus gene expression is not prompted by in vitro iron limitation, acid, alkaline, anaerobic or oxidative stress. However, subtelomeric gene expression is favoured following ex vivo neutrophil exposure and in comparative analyses of richly and poorly nourished laboratory cultured germlings. We found remarkable concordance between the A. fumigatus host-adaptation transcriptome and those resulting from in vitro iron depletion, alkaline shift, nitrogen starvation and loss of the methyltransferase LaeA. This first transcriptional snapshot of a fungal genome during initiation of mammalian infection provides the global perspective required to direct much-needed diagnostic and therapeutic strategies and reveals genome organisation and subtelomeric diversity as potential driving forces in the evolution of pathogenicity in the genus Aspergillus.

Figure 1. Comparative time-course of A. fumigatus Af293 germination and hyphal development in the murine lung, and laboratory culture.

(A) Time-course of Af293 germination and hyphal development in the neutropenic murine lung. (B) Microscopic appearance of Af293 germlings recovered from a typical single murine BALF, (harvested at 12–14 hours post-infection). (C) Microscopy of developmentally matched laboratory cultured Af293 germlings, following liquid culture for 12 hours in YPD at 37°C.

Figure 2. Correlation of log₂ ratios resulting from comparative transcriptional analysis of the laboratory cultured A. fumigatus cell populations T0 and T60 under varying mRNA amplification protocols.

Correlation of technically duplicated log₂ ratios between competitive hybridisations using single (aRNA₁), double (aRNA₂) and unamplified (totRNA) RNA samples. (A and B) Correlation between log₂ ratios obtained using cDNA derived from amplified and total RNA (totRNA v aRNA_r1 r = 0.74–0.80, totRNA v aRNA_r2 r = 0.74–0.80) (C) Cross-protocol pairings revealed highest correlations between slides using cDNA derived from amplified RNA (aRNA_r1 v aRNA_r2 r = 0.88–0.91) Surprisingly, technical replicates of slides using cDNA derived from total RNA (totRNA r = 0.80) were comparable to cross-protocol pairings (data not shown).

Figure 3. A genome-wide transcriptional snapshot of A. fumigatus Af293 during intiation of murine infection.

Red and green vertical lines correspond to individual up- and down-regulated genes, respectively. Thin light gray vertical lines indicate the positions of all other genes. (SM) and (asp_core) are density graphs of secondary metabolite and Aspergillus-core genes, respectively, expressed as a percentage of the total bases contained per gene type, per non-overlapping 2 kb of chromosomal sequence. Induced and repressed gene clusters, are depicted by red and green rectangles, respectively, below each chromosome. A complete listing of genes housed in these co-regulated clusters can be found in Table S6. Light blue/gray vertical bars represent putative centromeres and the pink vertical bar in chromosome 4 represents a region of ribosomal DNA.

Figure 4. Distribution of lineage specific and telomere-proximal genes among differentially expressed host adaptation dataset.

(A) Lineage specificity of A. fumigatus genes having altered transcript abundances, relative to laboratory culture, in the murine lung. The Aspergillus-core (Asp-core) set contains A. fumigatus Af293 proteins that have orthologues in A. clavatus (AAKD00000000), N. fischeri (AAKE00000000), Aspergillus terreus NIH2624 (AAJN01000000), Aspergillus oryzae RIB40, A. nidulans FGSC A4 and Aspergillus niger CBS 513.55 The Affc-core set were defined as A. fumigatus Af293 proteins that have ortholouges in N. fischeri and A. clavatus. The Affc-unique set is a sub-set of Affc-core proteins that do not have ortholouges in A. terreus, A. oryzae, A. nidulans or A. niger. Asterisks indicate gene sets which are listed in Table S5. Underlined values significantly deviate from the null hypothesis that an equal number of induced and repressed genes will occur in each cohort, as estimated by Chi-square analysis (Table 3). (B) Chromosomal distribution of A. fumigatus genes having altered transcript abundances, relative to laboratory culture, in the murine lung. Distances from telomeres (kb) are noted above pie charts. Asterisked gene sets are listed in Supplementary Table S5. Underlined values significantly deviate from the null hypothesis that an equal number of induced and repressed genes will occur in each cohort, as estimated by Chi-square analysis (Table 3).

Figure 5. Overlap between murine adaptation and in vitro stress datasets.

Venn diagrammatic representation of overlap between murine adaptation dataset and those of nitrogen starvation, iron starvation and alkaline shift. Genes are listed in Dataset S3.

Figure 6. Comparative analysis of A. fumigatus gene expression datsets.

A pan-experimental comparison of A. fumigatus gene expression aligning log₂ ratios obtained during host adaptation (mice); exposure to neutrophils (neut), increased expression in parental strain versus ΔlaeA mutant, acid shift (acid), iron starvation (iron), oxygen depletion (anaer) and oxidative stress (H₂O₂) for various genes. The colour bar indicates the range of log₂ expression ratios, grey bars indicate genes from which signals were undetectable for technical reasons. Experimental conditions are described in Materials and Methods. LaeA dataset is taken from Perrin et. al. . Comparative analyses were implemented in TM4 http://www.jcvi.org/cms/research/software/.

Figure 7. Characterisation of A. fumigatus growth, relative to YPD.

(A) Comparative analysis of Af293 radial growth on YPD and synthetic murine lung tissue medium (MLT). Triplicated, spot-inoculated plates containing single 100 spore inocula were incubated at 37°C. (B) Growth curve analysis of Af293, performed in triplicate using liquid YPD, or AMM containing 1% glucose and either 5 mM ammonium tartrate or 5 mM hyroxyproline as nitrogen source. Cultures were inoculated to a final concentration of 5×10⁶ spores/ml and incubated under aerobic conditions at 37°C with shaking at 150 rpm. At selected timepoints mycelia were harvested on Miracloth, encased in Whatmann paper and dried at 37°C for 48 hours before weighing.

Figure 8. Expression of LaeA-regulated genes during initiation of murine infection.

Venn diagram representation of overlap between genes repressed in ΔLaeA and those having increased transcript abundance during murine infection, according to proportions having subtelomeric locations, and secondary metabolism functionality (on the basis of annotation).